Objective:

Our objective is to develop robust three-dimensional (3D) organotypic culture models (OCMs) using human mesenchymal stem cells (MSCs) to examine critical phenomena of embryonic limb development and its susceptibility to perturbation by known teratogens and environmental toxicants. Our specific aims are: (1) to develop OCMs that recapitulate chondrogenic differentiation, subsequent chondrocyte maturation/hypertrophy, and finally cartilage segmentation; (2) validate these OCMs using their responses to known teratogens; (3) use these OCMs to evaluate chemicals from the TSCA inventory with suspected limb teratogenic activity; (4) couple our limb development OCMs to an upstream liver OCM to evaluate the role of hepatic metabolism and metabolic activation of xenobiotics on limb teratogenesis; and (5) assess species concordance at the OCM level by comparing results of toxicant exposure in OCMs using human MSCs versus rat embryonic limb bud mesenchymal cells.

Approach:

We will develop two limb development OCMs: one using a precartilage disc (CD) of MSCs to recapitulate chondrogenic differentiation and subsequent chondrocyte maturation/hypertrophy; and a second using a hyaline cartilage rod of MSCs that is subjected to mechanical activation to model the initiation of cartilage segmentation. The OCMs will be maintained in microbioreactors, custom designed by our VPROMPT collaborators in Project 5 to enable both perfusion-controlled exposure to candidate toxicants/teratogens and in situ mechano-activation. The spatiotemporal and dosimetric responses of OCMs will be monitored for specific cellular and morphogenetic activities using non-invasive live imaging of OCMs carrying AAV6 promoter-reporter constructs. Reporters will be chosen to match bioactivities associated with limb development, including cell adhesion, differentiation, matrix production, hypertrophy, matrix turnover, apoptosis, and cartilage segmentation. We will also assess OCM production of specific growth factors, cytokines, and/or metabolites using ion mobility mass spectrometry (IM-MS) and immunochemical measurements. Finally, at the end of the culture period, the phenotype of the OCMs will be assessed by histological, biochemical, and gene expression (RT-PCR) analyses.

Expected Results:

We expect to develop and validate two classes of OCMs using human MSCs for investigating toxicant exposure effects on chondrogenic steps in limb development. We expect these OCMs to be sufficiently robust to be useful for medium throughput toxicity screening and experimentally accessible to enable elucidation of Adverse Outcome Pathways.

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.